The Inner Mongolian pastoral region is one of the most important sheep meat production areas in China, playing a critical role in increasing herders’ income and supporting rural revitalization in border regions. Approximately 70% of local sheep are still managed under traditional year-round grazing systems, in which animal health and productive performance are strongly influenced by seasonal climatic variation and fluctuations in forage supply. With the continued expansion of the meat sheep industry, the natural grasslands of Inner Mongolia are increasingly subjected to ecological pressure arising from overstocking and accelerated pasture degradation [1].

As a unique digestive organ in ruminants, the rumen harbors a complex microbial ecosystem composed of bacteria, fungi, and archaea. Through the degradation of fibrous feed components, this microbial community provides more than 70% of the host’s metabolic energy, and its homeostasis is fundamental to the productive potential of sheep. However, under traditional grazing systems, forage yield and crude protein content decline markedly during winter and spring, which often leads to nutritional imbalance in grazing sheep and disrupts the stability of the rumen microbial community.

To elucidate the regulatory effects of seasonal variation on the rumen microbiota of grazing Mongolian sheep and to better understand digestive and metabolic patterns in grassland-based meat production systems, this study used Sunite ewes as the experimental model. The temporal dynamics of rumen microbial communities across different seasons were characterized, with the aim of improving the rumen microbiome reference dataset and providing a theoretical basis for the development of precise supplementary feeding strategies for grazing sheep during winter and spring periods.

The study site was located in Shangdu Town, Zhenglan Banner, Xilingol League, Inner Mongolia. Six 3-year-old Sunite ewes with similar body weights were randomly selected from a local grazing household as experimental animals. The sheep were maintained under a traditional year-round grazing system without any supplemental feeding.

Rumen fluid samples were collected at the middle of four representative seasons, namely winter (February), spring (May), summer (August), and autumn (October). Prior to morning grazing, approximately 10 mL of rumen fluid was obtained from each experimental ewe via external collection. The samples were filtered through four layers of sterile gauze and divided into three aliquots, which were then transferred into 5 mL sterile centrifuge tubes and immediately snap-frozen in liquid nitrogen, followed by storage at −80 °C until further analysis.

For each sampling period, rumen fluid was collected continuously for five consecutive days, and samples were subsequently pooled. From the mixed sample, 1 mL was used for microbial DNA extraction, which was then subjected to rumen microbial community analysis.

The results indicated that seasonal variation had no significant effect on ruminal bacterial richness in grazing sheep; however, microbial diversity was significantly higher in winter and spring than in summer. At the phylum level, the relative abundance of Firmicutes increased markedly in summer, whereas that of Bacteroidetes decreased significantly.

Seasonal changes did not significantly affect the alpha diversity of ruminal archaea in grazing sheep. Across all samples, approximately 99% of archaeal sequences were assigned to the phylum Euryarchaeota. At the genus level, Methanobrevibacter represented the dominant archaeal taxon, with higher abundance observed in autumn and winter compared with spring and summer, whereas the secondary dominant genus, Methanomassiliicoccus, exhibited an opposite seasonal pattern in relative abundance.

For ruminal fungi, diversity in May was significantly lower than that observed in the other sampling periods. Ascomycota remained the overwhelmingly dominant fungal phylum throughout all seasons, with its predominance remaining stable. At the genus level, the relative abundances of Preussia, Simplicillium, and Cryptococcus consistently showed a seasonal pattern characterized by lower abundance in spring and summer and higher abundance in autumn and winter.

This study confirmed that seasonal variation in forage nutrient composition and biomass production in natural grasslands is a primary driver shaping the succession of rumen microbial communities in grazing sheep. Bacterial alpha diversity was relatively lower in summer, while the Firmicutes-to-Bacteroidetes ratio peaked during summer, suggesting enhanced energy-harvesting efficiency under conditions of improved forage quality. Actinobacteria were more abundant in winter and are potentially involved in host immune modulation, whereas seasonal fluctuations in Proteobacteria likely reflect the combined effects of climatic stress and forage variability, indicating that core functional taxa can dynamically adapt to seasonal shifts in dietary structure.

Archaea were consistently dominated by Euryarchaeota, with two major methanogenic lineages exhibiting niche differentiation and symbiotic interactions with fibrolytic bacteria. Fungal communities varied in response to fluctuations in plant fiber content, with cold-season-adapted functional taxa being more enriched under low-temperature conditions.

In summary, bacteria, archaea, and fungi collectively contribute to maintaining ruminal functional stability in grazing sheep, enabling metabolic homeostasis across seasonal environmental transitions